James P, Wyss M, Lutsenko S, Wallimann T, Carafoli E
Laboratory of Biochemistry, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zürich.
FEBS Lett. 1990 Oct 29;273(1-2):139-43. doi: 10.1016/0014-5793(90)81069-z.
The ATP binding site of mitochondrial creatine kinase from chicken heart has been studied by modifying the purified enzyme with a 14C-labelled ATP analogue, C1RATP, in which the reactive label was covalently bound to the gamma-phosphate group of ATP. The modified enzyme was digested by pepsin, and a single radioactive nonapeptide was isolated by HPLC. Amino acid analysis and direct sequence determination revealed that the isolated peptide corresponds to amino acids 335-343 within the C-terminal region of Mi-CK, this peptide being highly preserved throughout evolution. Asp-335 is very likely the site of modification by C1RATP. The specificity of the ATP analogue for the active site of creatine kinase was demonstrated by the inhibition of the enzymatic activity of Mi-CK by C1RATP and by the prevention of this inhibition bij ADP.
通过用一种14C标记的ATP类似物C1RATP修饰纯化的鸡心线粒体肌酸激酶，对其ATP结合位点进行了研究。在C1RATP中，反应性标记物共价连接到ATP的γ-磷酸基团上。用胃蛋白酶消化修饰后的酶，通过高效液相色谱法分离出一种单一的放射性九肽。氨基酸分析和直接序列测定表明，分离出的肽对应于线粒体肌酸激酶（Mi-CK）C末端区域内的氨基酸335 - 343，该肽在整个进化过程中高度保守。天冬氨酸-335很可能是C1RATP的修饰位点。C1RATP对肌酸激酶活性位点的ATP类似物特异性通过C1RATP对Mi-CK酶活性的抑制以及ADP对这种抑制的预防得以证明。
